Preparation Of Microarrays Research Articles

Peptide microarrays for the determination of protease substrate specificity.

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.

TWO EFFICIENT POLYMERIC CHEMICAL PLATFORMS FOR OLIGONUCLEOTIDE MICROARRAY PREPARATION

ABSTRACT In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step with appropriate silanes (γ-aminopropyltriethoxysilane-APTES or 3-glycid-oxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.

Use of inexpensive dyes to calibrate and adjust your microarray printer.

A critical factor in the preparation of cDNA microarrays is the calibration and adjustment of the array printer to optimize spotting. Here are a couple techniques to help you detect the quality of your print, the proper calibration, and the condition of your printing pins. A simple way to determine the quality of the array spots is to examine the salt deposits remaining after the droplet of printer material has dried. However, this method does not necessarily reflect the morphology of the nucleic acid on the array because of the drying effects. Another common method includes the use of free Cy3-dCTP/dUTP or Cy5dCTP/dUTP dyes (Amersham Pharmacia Biotech, Piscataway, NJ, USA) or dyes containing oligonucleotides, followed by fluorescence scanning (1). Here, we describe an inexpensive alternative to using expensive fluorescent probes for the calibration and adjustment of pin-type array printers. Using a Stanford cDNA microarray printer (http://cmgm.stanford.edu/pbrown/ mguide/index.html) with ChipMaker 3 spotting pins (TeleChem International, Sunnyvale, CA, USA), we compared the results of printing under two conditions, with and without DNA. The goal of the calibration and adjustment steps is to obtain uniform and consistent spots. We used plain red and blue food coloring dye in 1:1000 dilution of 3× standard saline citrate (SSC) with sheared salmon sperm DNA at 0.25 mg/mL to mimic the actual printing conditions. All fluorescent scans were performed on a Packard/GSI Lumonics 3000 scanner (GSI Lumonics, Northville, MI, USA), with 85% laser and 90% PMT settings (Figure 1). The quality of the DNA spots was further examined with DNA binding dyes (data not shown). In the future, with the help of food coloring dye, we are going to compare different types of printing buffers such as 50% dimethyl sulfoxide (DMSO) with and without DNA, formamide, and a new printing buffer that recently became available from Mosaic Technologies (Waltham, MA, USA) and Clontech Laboratories (Palo Alto, CA, USA). REFERENCES

Preparation of DNA and protein micro arrays on glass slides coated with an agarose film.

A thin layered agarose film on microscope slides provides a versatile support for the preparation of arrayed molecular libraries. An activation step leading to the formation of aldehyde groups in the agarose creates reactive sites that allow covalent immobilization of molecules containing amino groups. Arrays of oligonucleotides and PCR products were prepared by tip printing. After hybridization with complementary fluorescence labeled nucleic acid probes strong fluorescence signals of sequence-specific binding to the immobilized probes were detected. The intensity of the fluorescence signals was proportional to the relative amount of immobilized oligonucleotides and to the concentration of the fluorescence labeled probe. We also used the agarose film-coated slides for the preparation of protein arrays. In combination with specific fluorescence labeled antibodies these protein arrays can be used for fluorescence linked immune assays. With this approach different protein tests can be performed in parallel in a single reaction with minimal amounts of the binding reagents.

Immobilization of Oligonucleotides onto a Glass Support via Disulfide Bonds: A Method for Preparation of DNA Microarrays

The covalent attachment of disulfide-modified oligonucleotides to a mercaptosilane-modified glass surface is described. This method provides an efficient and specific covalent attachment chemistry for immobilization of DNA probes onto a solid support. Glass slides were derivatized with 3-mercaptopropyl silane for attachment of 5-prime disulfide-modified oligonucleotides via disulfide bonds. An attachment density of approximately 3 × 105oligonucleotides/μm2was observed. Oligonucleotides attached by this method provided a highly efficient substrate for nucleic acid hybridization and primer extension assays. In addition, we have demonstrated patterning of multiple DNA probes on a glass surface utilizing this attachment chemistry, which allows for array densities of at least 20,000 spots/cm2.